| Spikelet is a special inflorescence structure of grass plants such as rice,which can produce one or more florets/grains directly,so it plays a very important role in the formation of grain number per panicle of several important gramineous crops.The spikelet of rice displays a unique structure compared with others.In the past 20 years,a large number of studies have been carried out about the flower/panicle development in rice,including flower initiation,branch development,and floret development.However,the research on the mechanism of spikelet development is just started,and there are many problems need to be solved.In order to establish molecular network of spikelet development regulation,and provide theoretical basis for rice molecular breeding work,we need to explore more correlated mutants,clone the genes,further study their functions and mechanisms.In previous studies,we have identified two genes,NONSTOP GLUMES 1(NSG1)and STAMLESS 1(SL1),which involved in spikelet and floral organ development in rice.Both two genes encode the conserved QALGGH C2H2 zinc finger transcription factors in rice.Through phenotype analysis,evolution analysis and expression analysis of the two genes,we preliminarily analyzed the function of NSG1 gene to affect the characteristic of lateral organs of spikelet,and the function of SL1 gene to affect the characteristic of inner organs of floret,such as lodicule and stamen.In this paper,we further revealed the in-depth molecular mechanism of the two C2H2 zinc finger transcription factors encoded by NSG1 and SL1 in regulating the development of spikelet/floret in rice,which provided new gene resources for molecular breeding of "three-florets spikelet" and constructed a mature floral organ specification gene regulatory network(FOS-GRN),the main conclusions are as follows:1.Molecular mechanism of NSG1 gene regulating spikelet developmentThe spikelet is an inflorescence structure unique to grasses and is one of the most important elements affecting quality and yield of rice.In rice,the spikelet contains one fertile terminal floret(consisting of four whorls of floral organs: one lemma and one palea,two lodicules,six stamens,and one pistil),one pair of sterile lemmas,and one pair of rudimentary glumes.The hypothesis of " three-florets spikelet " suggests that the spikelet of rice ancestors contained a terminal floret and two lateral florets.According to the proposal,the rice spikelet is a ―three-florets spikelet‖ and the lateral florets degenerated into sterile lemmas during evolution.In 2017,we identified a gain-of-function mutant lf1.The gain of function of LF1 results in development of a lateral floret(including the palea,lodicule,stamens,and pistil)in the axil of the sterile lemma,providing direct evidence for the hypothesis of " three-florets spikelet".However,there are still some problems to be solved in order to restore the original " three-florets spikelet ",such as the degenerated glume needs to be restored to lemma,so as to protect the inner flower organs together with the lemma.In the study,we characterized three allelic recessive mutants in rice(Oryza sativa),nonstop glumes 1-1(nsg1-1),nsg1-2,and nsg1-3.In these mutants,organs including rudimentary glume,sterile lemma,palea,lodicule,and filament were elongated and/or widened,or transformed into lemma-and/or mrp-like organs.NSG1 encoded a member of the C2H2 zinc finger protein family and was expressed in the whole spikelet primordium at the early stage of inflorescence development.In this study,by using a variety of approaches referring to genetics,cytology and molecules,the molecular mechanism of NSG1 regulating the development of lateral organs was deeply explored.The main conclusions are as follows:1.The role of NSG1 in specification of sterile lemmas and rudimentary glumesIn three allelic mutants of NSG1,the rudimentary glumes and the sterile lemmas were elongated and/or widened,and most of the rudimentary glumes and a minority of the outer sterile lemmas were transformed into the lemma and/or mrp-like organ,and most of outer sterile lemmas and inner sterile lemmas were transformed into the lemma-like organ,suggesting that NSG1 are involved in maintaining the identity of rudimentary glumes and/or sterile lemmas by preventing incorrect cell differentiation.2.NSG1 regulates palea and lodicule developmentIn the nsg1-1 mutant,over 50% of spikelets contained a lemma-like palea as the mrp gained a lemma-like identity to various degrees,and approximately one-third of spikelets showed a degenerated palea owing to the degeneration or absence of the bp.These results indicate that NSG1 plays a dual role in regulating of both bp and mrp development in the palea,possibly through different regulation and control patterns.3.NSG1 maintains organ identity of the spikelet by regulation of LHS1,MFO1,DL,and G1 expressionIn the nsg1-1 spikelet,LHS1,DL,and MFO1 were ectopically expressed in two or more organs,including the rudimentary glume,sterile lemma,palea,lodicule,and stamen.We further verified that the NSG1 protein could bind to the sites in the promoter of LHS1,interact with rice co-repressors Os TRPs,and then recruit HDACs to mediate the histone acetylation level of LHS1.2.Molecular mechanism of SL1 gene regulating floral organ developmentOver the past three decades,molecular and genetic mechanisms of flower development have been studied intensively in both monocotyledons and dicotyledons,particularly in Arabidopsis(Arabidopsis thaliana).However,in rice and other graminaceous species,an equivalent FOS-GRN network remains largely unknown.The sl1 mutant has elongated lodicules in whorl 2 same with spw1 mutant.The stamens in whorl 3 of both spw1 and sl1 mutants were transformed into pistil or pistil-like organs.Through expression pattern analysis of SPW1 and DL in sl1,we found that expression of DL gene extended to the third whorl,and the expression of SPW1 was significantly reduced.It is speculated that the loss function of SL1 leads to the ectopic expression of DL gene and the reduction of SPW1,which is similar to the pistilloid-stamen in spw1 mutant.By analysing the phenotypes and patterns of gene expression in single and double mutants,and using a variety of molecular/genetic methods,we proposed a gene regulatory network for floral organ development in rice,SL1–SPW1–DL,in which SL1 directly activates SPW1 expression,and SPW1 then inhibits ectopic expression of DL in the stamen and the lodicule.In addition,our datas further suggested that SL1 is able to interact with the Os Ruv B1-related Os Tip60 chromatin-remodeling complex to mediate the H4 histone modification on SPW1 gene.1.SL1 was responsible for activation of SPW1 but not repression of DL.In the sl1 mutant,expression of SPW1 was barely detected in the precursor cells or primordia of lodicules and stamens.However,expression of SPW1 remained extremely weak in whorls 2 and 3 in the sl1+dl double mutant,similar to that observed in the sl1 single mutant.Thus,low-level expression of SPW1 in the sl1 mutant was not caused by ectopic expression of DL,but loss-of-activation of SL1.Therefore,SL1 was responsible for activation of SPW1 but not repression of DL.2.SL1 was able to bind to the promoter of SPW1To clarify whether SL1 directly regulates SPW1 and the associated mechanism,we conducted the Ch IP-q PCR analysis and the dual-luciferase reporter assay to investigate potential interaction proteins and activating mechanism.The results suggested that SL1 may directly regulate SPW1 expression by binding to the SPW1 promoter.3.SL1 being able to interact with chromatin-remodeling complex Tip60(containing Os Ruv B1,Os DP1/E2 F and Os Tip60 proteins)to mediate the histone H4 modification on SPW1 gene.SL1 is a nuclear-located transcription factor but shows no activation/repression activity.In order to explore the in-depth molecular mechanism of SL1 activating SPW1,two chromatin remodeling complex related proteins,Os Ruv B1 and Os DP,were screened out by Co-IP and using a point to point yeast two hybrid system.Os Ruv B1 is an important component of chromatin remodeling complexes Tip60 / Ino80 / Swr,while Os DP can recruit Tip60 complex through E2 F transcription factors participating in many physiological processes.These results suggested that SL1 being able to interact with chromatin-remodeling complex Tip60(containing Os Ruv B1,Os DP1/E2 F and Os Tip60 proteins)to activate the SPW1 gene. |
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